The immune system, by and large, plays a primary role to minimize and/or prevent infection, in general, and periodontal disease, in particular. We propose that immunoregulatory abnormalities contribute to the pathogeneisis of and susceptibility to periodontal disease. The fundamental hypothesis of our studies is that periodontal pathogens produce immunosuppressive proteins (ISP) that mediate local and/or systemic immunosuppression, thereby enhancing their own virulence and/or that of other opportunistic microorganisms. In this regard, our investigations have demonstrated that Actinobacillus actionmycetemcomitans produces an ISP that impairs human T- and B-cell function. We plan to focus this investigation on the A. actinomycetemcomtians ISP which we have shown to be a product of the cytolethal distending toxin (Cdt) operon. We have demonstrated that Cdt acts by inducing cell cycle arrest of human T-cells in the G2 phase which eventually leads to cell death via activation of the apoptotic cascade. More recently, we have demonstrated that the A. actinomycetemcomitans Cdt functions as an AB2 toxin where CdtB is the active (A) unit and the complex of CdtA and CdtC comprise the binding (B) unit. Furthermore, we have demonstrated that membrane lipid microdomains and signaling pathways involving protein kinase C (PKC) activation are critical to the mode of action of the toxin. The objectives of this proposal are: (1) define the role of membrane lipid microdomains in relation to the cascade of events responsible for toxin-induced G2 arrest and cell death;(2) to determine the relationship between CdtB structure and function. The study is divided into 4 specific aims: (1) To determine if Cdt holotoxin interaction with host cell plasma membranes requires cholesterol;(2) To determine the relationship between membrane microdomains, cellugyrin and CdtB internalization;(3) To determine the role of PKC activation and its relationship to membrane microdomains in Cdt-induced G2 arrest and apoptosis;and (4) To determine if CdtB exhibits phosphatidylinositol (PI) phosphatase activity and, if so, determine if the activity is critical to the toxins mode of action. It is anticipated that these studies will lead to a more detailed understanding of Cdt and provide: (1) important insight into the pathogenesis of disease caused by Cdt-producing bacteria and (2) a rationale on which therapeutic intervention may be developed to prevent or limit disease.